Multidrug resistance of infectious bacterial pathogens has emerged as one of the greatest human health problems. As such, there is a great impetus to develop new generation antibiotics. Cationic amphipathic antimicrobial peptides (AMPs) are nature's ancient antibiotics. They possess a broad spectrum of antimicrobial activity. However, AMP based antibiotics have short lifetime due to protease degradation. Peptide mimics, the so-called peptidomimetics, were recently conceptualized and explored in hope of identifying antibiotic substitutes with superior antimicrobial properties. We are at the nexus of this effort. In particular, we have developed a new class of peptidomimetics referred to as ?-AApeptides, which are named after the N-acylated- N-aminoethyl amino acid unit. ?-AApeptides outperform natural AMPs by providing better potency, and excellent resistance to protease degradation. They are also not prone to eliciting bacterial resistance. Another advantage of ?-AApeptides is their easy accommodation of versatile functional groups. This enables us to design and synthesize various types of ?-AApeptides. So far we have developed a few subclasses of ?- AApeptides through cyclization and lipidation. It was found that ?-AApeptides primarily target bacterial membranes. To translate ?-AApeptides into clinically efficient and safe antibiotic substitutes, a detailed mechanistic understanding of ?-AApeptides interacting with bacterial membranes becomes a prerequisite. For this proposal our first specific aim is to determine interactions between ?-AApeptides and model membranes mimicking bacterial cytoplasmic and outer membranes. Our biophysical studies will elucidate (1) bacterial membrane properties (i.e., structure and nanomechanics) with different lipid compositions, (2) bacterial lipid effect in dictating ?-AApeptide activity, and (3) structural and compositional characteristics of ?-AApeptides that govern their antimicrobial activity. Our second specific aim is to study morphology and elasticity of live bacterial cells modulated by ?-AApeptides. The whole-cell studies will unveil (1) multiple stages employed by ?- AApeptides in killing bacteria, ranging from cell envelope alteration to complete lysis, and (2) dynamic impact of ?-AApeptides on mechanical integrity of live bacterial cells. Bacterial membrane interactions emerging from molecular and cellular level studies will be integrated into our third specific aim, which is to develop potent ?- AApeptides specifically targeting Gram-positive methicillin-resistant S. aureus (MRSA) and Gram-negative P. aeruginosa with minimum inhibitory concentrations meeting clinical standards. The innovation of the proposed project is to establish a platform maximizing the expertise of biophysicists and biochemists. We believe a detailed mechanistic understanding discerned from our biophysical studies will substantially benefit structure and function optimization in generating novel ?-AApeptides. The results from the proposed project will have a strong impact on rational design of novel peptidomimetics with larger antimicrobial efficacy and less propensity of eliciting bacterial resistance.